Summary of study ST001073

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000718. The data can be accessed directly via it's Project DOI: 10.21228/M8VT2P This work is supported by NIH grant, U2C- DK119886.

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Study IDST001073
Study TitleLipid profiling of Wnt3a-induced optic nerve regeneration
Study Typeuntargeted lipid profiling
Study SummaryWe analyzed lipid profiles of mouse retina and optic nerve for 2 time points - 7 and 15 days post-crush, and 3 conditions - intact control, optic nerve crush + vehicle (saline) intravitreal injection, optic nerve crush + 20 ng of Wnt3a injection.
Institute
University of Miami
DepartmentOphthalmology, Bascom Palmer Eye Institute
LaboratorySanjoy K. Bhattacharya lab
Last NameBhattacharya
First NameSanjoy
AddressBascom Palmer Eye Institute (McKnight Bldg.), 1638 NW 10th Avenue, Suite 707A, University of Miami, Miami, FL, 33136
Emailsbhattacharya@med.miami.edu
Phone305-482-4103
Submit Date2018-10-03
Num Groups6
Total Subjects12
Raw Data AvailableYes
Raw Data File Type(s).raw
Analysis Type DetailLC-MS
Release Date2018-12-11
Release Version1
Sanjoy Bhattacharya Sanjoy Bhattacharya
https://dx.doi.org/10.21228/M8VT2P
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000718
Project DOI:doi: 10.21228/M8VT2P
Project Title:Lipid profiling of Wnt3a-induced optic nerve regeneration
Project Type:untargeted lipid profiling
Project Summary:We analyzed lipid profiles of mouse retina and optic nerve at 2 time points - 7 and 15 days post-crush, and 3 conditions - intact control, optic nerve crush + vehicle (saline) intravitreal injection, optic nerve crush + 20 ng of Wnt3a injection.
Institute:University of Miami
Department:Ophthalmology, Bascom Palmer Eye Institute
Laboratory:Sanjoy K. Bhattacharya lab
Last Name:Bhattacharya
First Name:Sanjoy
Address:Bascom Palmer Eye Institute (McKnight Bldg.), 1638 NW 10th Avenue, Suite 707A, University of Miami, Miami, FL, 33136
Email:sbhattacharya@med.miami.edu
Phone:305-482-4103
Contributors:Abigail S. Hackam, Tal Carmy, Anna Trzeciecka

Subject:

Subject ID:SU001117
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:Tcf/LacZ mice
Age Or Age Range:6-8 weeks
Gender:Male and female
Animal Inclusion Criteria:The Tcf/LacZ strain was previously backcrossed onto the C3H background C3H background, and confirmed by PCR to lack the rd1 mutation

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Crush Saline treatment Wnt3a treatment Day post-crush
SA072409QC2N/A N/A N/A 15
SA072410QC1N/A N/A N/A 15
SA072411QC4N/A N/A N/A 15
SA072412QC3N/A N/A N/A 15
SA072413QC6N/A N/A N/A 15
SA072414QC5N/A N/A N/A 15
SA072415HA2_14No No No 15
SA072416HA2_5No No No 15
SA072417HA2_2No No No 15
SA072418HA2_15No No No 15
SA072419HA2_3No No No 15
SA072420HA2_24No No No 15
SA072421HA2_1No No No 15
SA072422HA2_23No No No 15
SA072423HA2_18No No No 15
SA072424HA2_17No No No 15
SA072425HA2_16No No No 15
SA072426HA2_4No No No 15
SA072427HA_16No No No 7
SA072428HA_11No No No 7
SA072429HA_12No No No 7
SA072430HA_15No No No 7
SA072431HA_20No No No 7
SA072432HA_8No No No 7
SA072433HA_7No No No 7
SA072434HA_3No No No 7
SA072435HA_4No No No 7
SA072436HA_23No No No 7
SA072437HA_19No No No 7
SA072438HA_24No No No 7
SA072439HA2_12Yes No Yes 15
SA072440HA2_11Yes No Yes 15
SA072441HA2_13Yes No Yes 15
SA072442HA2_7Yes No Yes 15
SA072443HA2_8Yes No Yes 15
SA072444HA2_9Yes No Yes 15
SA072445HA_9Yes No Yes 7
SA072446HA_10Yes No Yes 7
SA072447HA_1Yes No Yes 7
SA072448HA_2Yes No Yes 7
SA072449HA_6Yes No Yes 7
SA072450HA_5Yes No Yes 7
SA072451HA2_22Yes Yes No 15
SA072452HA2_21Yes Yes No 15
SA072453HA2_10Yes Yes No 15
SA072454HA2_6Yes Yes No 15
SA072455HA2_19Yes Yes No 15
SA072456HA2_20Yes Yes No 15
SA072457HA_21Yes Yes No 7
SA072458HA_14Yes Yes No 7
SA072459HA_22Yes Yes No 7
SA072460HA_18Yes Yes No 7
SA072461HA_17Yes Yes No 7
SA072462HA_13Yes Yes No 7
Showing results 1 to 54 of 54

Collection:

Collection ID:CO001111
Collection Summary:All procedures involving mice were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Care and Use Committee at the University of Miami. The optic nerve crush and associated treatments were done on Tcf/LacZ mice. As described previously1, the Tcf/LacZ mouse is a transgenic canonical Wnt reporter line that allows localization of active Wnt/β-catenin signaling. In these mice, binding of endogenous nuclear β-catenin to T-cell factor/lymphoid enhancer-binding factor (TCF/Lef) elements in a Wnt-responsive enhancer/promoter region upstream of the LacZ transgene leads to induction of LacZ/β-galactosidase (β-gal) expression wherever Wnt signaling is active. The Tcf/LacZ strain was previously backcrossed onto the C3H background C3H background, and confirmed by PCR to lack the rd1 mutation. Optic nerve crush (ONC) injury was performed as described previously. A total of twelve Tcf/LacZ mice were used for this study. Six mice were used for a 7 day period and six mice were used for a 14 day period. The mice were used at the age of 6-8 weeks and were anesthetized using a ketamine/xylazine cocktail which was injected intraperitoneally. Once the mice were under the anesthesia, the eyes were locally anesthetized with 0.5% proparacaine hydrochloride. The mice were placed on a heating pad to begin the optic nerve crush procedure. Mice of either sex were randomly selected to receive intravitreal injection of recombinant 20ngWnt3a ligand group or saline control group (saline was injected with equivalent volume as recombinant 20ngWnt3a ligand). A small diabetic needle was used to make an incision in the superior posterior area of the conjunctiva-sclera border of the left eye. After which, either recombinant 20ngWnt3a ligand or saline was injected intravitreally using a 1.5 cm 33-gauge Hamilton needle (Hamilton Company, Reno, NV). The injection needle was angled to avoid hitting the lens. The surface membrane of the left eye was cut around the conjunctiva-sclera border. Dumont #5 forceps were inserted between the membrane and the globe to move the surrounding tissues while searching for the optic nerve. Once the optic nerve was located, the forceps’ teeth surrounded the nerve 1 mm from the globe and crushed the nerve for 5 seconds. The crush is successful if there is very minimal or no damage to the surrounding blood supply. Affected left eyes were treated with topical erythromycin ointment and 1mg/ml of Buprenorphine-SR lab was injected subcutaneously. Any mouse with excessive bleeding around the eye was excluded from the study. Mouse’s overall health and left eye’s health were monitored daily from day 1 post crush until day 7 post crush. Mice portraying lethargy 24 hours after surgery or eye infection to the affected eye days after surgery were excluded from the study. OCT scans were obtained from left and right eyes of the mouse while anesthetized on the day of euthanasia. Nerves and retinas were removed immediately after OCT scan for mass spectrometry analysis.
Sample Type:Eye tissue
Collection Frequency:7 and 15 days post-crush
Volumeoramount Collected:entire retina or optic nerve
Storage Conditions:-80℃
Storage Vials:low-binding 1.5 mL Eppendorf tubes

Treatment:

Treatment ID:TR001131
Treatment Summary:Mice of either sex were randomly selected to receive intravitreal injection of recombinant 20ng Wnt3a ligand group or saline control group (saline was injected with equivalent volume as recombinant 20ng Wnt3a ligand). A small diabetic needle was used to make an incision in the superior posterior area of the conjunctiva-sclera border of the left eye. After which, either recombinant 20ngWnt3a ligand or saline was injected intravitreally using a 1.5 cm 33-gauge Hamilton needle (Hamilton Company, Reno, NV). The injection needle was angled to avoid hitting the lens. The surface membrane of the left eye was cut around the conjunctiva-sclera border. Dumont #5 forceps were inserted between the membrane and the globe to move the surrounding tissues while searching for the optic nerve. Once the optic nerve was located, the forceps’ teeth surrounded the nerve 1 mm from the globe and crushed the nerve for 5 seconds. The crush is successful if there is very minimal or no damage to the surrounding blood supply. Affected left eyes were treated with topical erythromycin ointment and 1mg/ml of Buprenorphine-SR lab was injected subcutaneously. Animal Anesthesia: ketamine/xylazine cocktail intraperitoneally, eyes were locally anesthetized with 0.5% proparacaine hydrochloride.Any mouse with excessive bleeding around the eye was excluded from the study. Mouse’s overall health and left eye’s health were monitored daily from day 1 post crush until day 7 post crush. Animal Endpoint Clinical Signs: Mice portraying lethargy 24 hours after surgery or eye infection to the affected eye days after surgery were excluded from the study.
Treatment:optic nerve crush, intravitreal injection
Treatment Compound:Wnt3a or saline
Treatment Route:intravitreal injection
Treatment Dosevolume:20 ng of Wnt3a or equal volume of saline
Treatment Doseduration:single injection
Animal Anesthesia:See summary
Animal Endp Euthanasia:CO2 + decapitation
Animal Endp Tissue Coll List:retina, optic nerve
Animal Endp Clinical Signs:See summary

Sample Preparation:

Sampleprep ID:SP001124
Sampleprep Summary:4 mL of methanol (LC-MS grade) and 2 mL of chloroform (LC-MS grade) were added to each sample. After 2 min vigorous vortexing and 2 min sonication in ultrasonic bath, the samples were incubated at 48˚C overnight (in borosilicate glass vials, PTFE-lined caps). The following day, 2 mL of water (LC-MS grade) and 1 mL of chloroform were added, samples vigorously vortexed for 2 min and centrifuged at 3000 RCF, 4˚C for 15 min to obtain phase separation. Lower phases were collected and dried in centrifugal vacuum concentrator. Samples were stored at -20˚C until reconstituted in 150 µL of chloroform:methanol (1:1) prior to mass spectrometric analysis. Lipid profiling was performed in 2 batches: 7 day and 15 day period. For the 15 day period, quality control (QC) pooled sample was prepared and run 6 times throughout the batch.
Processing Method:lipid extraction
Processing Storage Conditions:Described in summary
Extraction Method:chloroform, methanol, water phase separation
Extract Storage:-80℃
Sample Resuspension:150 µL of chloroform:methanol (1:1)
Sample Spiking:no internal standards added

Combined analysis:

Analysis ID AN001755
Analysis type MS
Chromatography type Reversed phase
Chromatography system Thermo Accela 600
Column Acclaim C30 column: 3 µm, 2.1x150 mm (Thermo)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive Orbitrap
Ion Mode UNSPECIFIED
Units relative abundance (main area)

Chromatography:

Chromatography ID:CH001239
Chromatography Summary:Reversed phase chromatographic separation utilized Accela Autosampler (Thermo), Accela 600 pump (Thermo) and Acclaim C30 column: 3 µm, 2.1x150 mm (Thermo). The column temperature was maintained at 30˚C and tray temperature at 20˚C. Solvent A was composed of 10 mM ammonium acetate (LC-MS grade) in 60:40 methanol:water (LC-MS grade) with 0.2% formic acid (FA; LC-MS grade). Solvent B was composed of 10 mM ammonium acetate with 60:40 methanol:chloroform with 0.2% FA. The flow rate was 260 µL/min and injection volume was 10 µL. The gradient was 35-100% solvent B over 13.0 min, 100% solvent B over 13.0-13.8 min, 100-35% solvent B over 13.8-14.5 min, 35% solvent B over 14.5-18.0 min, 0% solvent B over 18.0-20.0 min.
Instrument Name:Thermo Accela 600
Column Name:Acclaim C30 column: 3 µm, 2.1x150 mm (Thermo)
Column Temperature:30˚C
Flow Gradient:The gradient was 35-100% solvent B over 13.0 min, 100% solvent B over 13.0-13.8 min, 100-35% solvent B over 13.8-14.5 min, 35% solvent B over 14.5-18.0 min, 0% solvent B over 18.0-20.0 min.
Flow Rate:260 µL/min
Sample Injection:10 µL
Solvent A:10 mM ammonium acetate (LC-MS grade) in 60:40 methanol:water (LC-MS grade) with 0.2% formic acid (FA; LC-MS grade)
Solvent B:10 mM ammonium acetate with 60:40 methanol:chloroform with 0.2% FA
Analytical Time:20 min
Washing Buffer:methanol
Chromatography Type:Reversed phase

MS:

MS ID:MS001623
Analysis ID:AN001755
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:positive and negative mode aligned together
Ion Mode:UNSPECIFIED
Capillary Temperature:310˚C (negative mode) or 350˚C (positive mode)
Collision Energy:15, 30, 45, 60, 75, 90 (in positive and negative mode separately; total 12 runs per sample)
Ion Spray Voltage:4.4 kV
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